The 26S proteasome complex is essential for targeted protein degradation. It is composed of a 20S core particle (CP) and a 19S regulatory particle (RP). The 20S CP confers the proteolytic activity to the proteasome. The 19S RP has six different ATPase components, and regulates the function of the 20S CP in an ATP-dependent manner. Intriguingly, several studies have implicated the non-proteolytic role of the 19S RP in transcriptional initiation or activation independently of the 20S CP. However, the detailed regulatory mechanisms of transcriptional initiation by the 19S RP remained largely unknown in living cells. Based on our current NIH R15 funding, we have demonstrated in yeast that the 19S RP, but not 20S CP, is recruited to the GAL1 UAS (upstream activating sequence), and establishes a specific interaction network of Gal4-SAGA- Mediator (SAGA, Spt-Ada-Gcn5-acetyltransferase) at the UAS by enhancing the interaction of activator Gal4 with coactivator SAGA, which promotes the formation of preinitiation complex (PIC) at the core promoter to initiate transcription. We subsequently found similar results at several other genes. Overall, we find that 19S RP promotes the interaction of activator with coactivator for facilitating the formation of PIC, and hence transcriptional initiation. However, it is not clearly understood how the 19S RP promotes the interaction of activator with coactivator. Further, the roles of different ATPases of the 19S RP in such regulation are yet unknown. Moreover, it is not known whether the 19S RP functions as a global regulator of transcriptional initiation. Answers to these important questions will significantly contribute to the regulatory roles and mechanisms of the 19S RP in transcriptional initiation. Therefore, in this NIH R15 renewal application, we propose to address these questions. We will specifically determine (i) the role of the 19S RP in global transcriptional initiation, and (ii) how the 19S RP or its ATPase activity promotes the activator-coativator interaction, and hence transcriptional initiation. We will address these specific research aims primarily using the ChIP (chromatin immunoprecipitation), mutational, transcriptional, biochemical, biophysical, and ChIP- seq methodologies. The outcomes of this research proposal will provide important information on the 19S RP regulation of eukaryotic transcriptional initiation, and hence will significantly contribute to our current understanding of eukaryotic gene regulation. Since the transcription machinery as well as the 19S RP is conserved throughout eukaryotes from yeast to humans, these results in yeast will provide important resources for research in humans. Thus, the results will assist in the development of transcription-based therapeutic agents, as a growing number of diseases including cancer are linked to aberrant transcriptional initiation and/or are characterized by altered patterns of gene expression.